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distinctive g protein-dependent signaling by protease-activated receptor 2 (par2) in smooth muscle: feedback inhibition of rhoa by camp-independent pka.we examined expression of protease-activated receptors 2 (par2) and characterized their signaling pathways in rabbit gastric muscle cells. the par2 activating peptide sligrl (par2-ap) stimulated gq, g13, gi1, pi hydrolysis, and rho kinase activity, and inhibited camp formation. stimulation of pi hydrolysis was partly inhibited in cells expressing par2 sirna, gaq or gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of g protein sign ...201323825105
evaluation of cp chromo select agar for the enumeration of clostridium perfringens from water.the european directive on drinking water quality has included mcp agar as the reference method for recovering clostridium perfringens from drinking waters. in the present study, three media (mcp, tscf and cp chromo select agar) were evaluated for recovery of c. perfringens in different surface water samples. out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for c. perfringens in at least one of the culture media. green co ...201323816139
comparative immunochemical characteristics of botulinum neurotoxin type a and its associated proteins.clostridium botulinum strains secrete their neurotoxins (bont) along with a group of neurotoxin-associated proteins (naps) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the g.i. tract. a major component of naps is hn-33, a 33 kda protein, which is also protease resistant and strongly protects bont. the complex form of bont/a is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic ...201323811077
the two-component system cbo2306/cbo2307 is important for cold adaptation of clostridium botulinum atcc 3502.clostridium botulinum is a notorious foodborne pathogen. its ability to adapt to and grow at low temperatures is of interest for food safety. two-component systems (tcss) have been reported to be involved in cold-shock and growth at low temperatures. here we show the importance of tcs cbo2306/cbo2307 in the cold-shock response of c. botulinum atcc 3502. the relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in t ...201323810493
transport of the botulinum neurotoxin-associating protein, nontoxic nonhemagglutinin, across the rat small intestinal epithelial cell monolayer.botulinum neurotoxin (bont) associates with nontoxic nonhemagglutinin (ntnha) yielding a complex in culture. bont and ntnha have similar domain organizations, implying that they share common functions, although this remains unclear. here, we examined cell monolayer transport of serotype d ntnha in the rat intestinal epithelial cell line iec-6. ntnha and bont both bound to the cell and were transported across the cell layer. ntnha contains a qxw motif and a β-trefoil fold, both common in sugar ch ...201323809018
regulation of toxin synthesis in clostridium botulinum and clostridium tetani.botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. botulinum neurotoxin (bont) associates with non-toxic proteins (antps) to form complexes of various sizes, whereas tetanus toxin (tent) does not form any complex. the bont and antp genes are clustered in a dna segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various clostridium botulinum types and subty ...201323769754
the effect of ph on growth of clostridium botulinum type a and expression of bonta and botr during different growth stages.in this study, the effects of ph on the growth, relative expressions of bonta and botr genes, and neurotoxin formation of foodborne pathogens clostridium botulinum type a were systematically studied throughout its growth stage. as in the previous reports, no c. botulinum growth was observed at extremely acidic ph. however, the effect of alkaline ph on the growth and neurotoxin production of c. botulinum was first revealed in this study. the maximum growth rate at ph 9.0 was similar to that at ot ...201323767855
botulism in children: a diagnostic dilemma in developing countries.botulism is a well-known disease of the neuromuscular junction. it is a rare but curable cause of paralysis in paediatric population. in addition to classical clinical signs and symptoms, the diagnosis of botulism requires laboratory confirmation of intoxication by various biological tests. these include demonstration of botulinum toxin in serum or isolation of the clostridium botulinum from stool/gastric aspirates. however, it is not always possible to confirm intoxication due to unavailability ...201323763811
validation of a real-time pcr based method for detection of clostridium botulinum types c, d and their mosaic variants c-d and d-c in a multicenter collaborative trial.two real-time pcr arrays based on the genedisc(®) cycler platform (pall-genedisc technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate clostridium botulinum types c, d and their mosaic variants c-d and d-c that are associated with avian and mammalian botulism. the genedisc(®) arrays developed as part of the dg home funded european project 'anibiothreat' were highly sensitive and specific when tested on pure isolates and natur ...201323669132
the type f6 neurotoxin gene cluster locus of group ii clostridium botulinum has evolved by successive disruption of two different ancestral precursors.genome sequences of five different group ii (nonproteolytic) clostridium botulinum type f6 strains were compared at a 50-kb locus containing the neurotoxin gene cluster. a clonal origin for these strains is indicated by the fact that sequences were identical except for strain eklund 202f, with 10 single-nucleotide polymorphisms and a 15-bp deletion. the essential topb gene encoding topoisomerase iii was found to have been split by the apparent insertion of 34.4 kb of foreign dna (in a similar ma ...201323645598
environmental factors influencing the prevalence of a clostridium botulinum type c/d mosaic strain in nonpermanent mediterranean wetlands.between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of mancha húmeda (central spain). these outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (oxyura leucoceophala). here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in july (always >26°c). the presence of clostridium botulinum type c/d in wetland sediments was de ...201323645197
clostridium botulinum strain af84 contains three neurotoxin gene clusters: bont/a2, bont/f4 and bont/f5.sanger and shotgun sequencing of clostridium botulinum strain af84 type af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. the three toxin gene clusters consisted of bont subtypes a2, f4 and f5. the bont/a2 and bont/f4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/f5 toxin gene cluster was located within a large 246 kb plasmid. these findings are the first ident ...201323637798
purification and characterization of neurotoxin complex from a dual toxin gene containing clostridium botulinum strain ps-5.botulinum neurotoxins are produced as a toxin complex (tc) which consists of neurotoxin (nt) and neurotoxin associated proteins. the characterization of nt in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. the presence of nt genes was validated by pcr amplification of toxin specific fragments from genomic dna of clostridium botulinum strain ps-5 which indicated the presence of both serotype a and b genes on ps-5 genome. further, ...201323625059
type c and c/d toxigenic clostridium botulinum is not normally present in the intestine of healthy broilers.toxigenic clostridium botulinum spores are widely distributed in wetland environments and are frequently recovered from healthy wild birds, where ingestion of toxic maggots from carcasses is considered a major initiating factor for botulism outbreaks. toxic carcasses can perpetuate an existing outbreak also in poultry, but their importance as an initiating factor for poultry botulism is less clear. although toxigenic c. botulinum can be recovered from healthy broilers during outbreaks, there are ...201323623689
development and characterization of six monoclonal antibodies to hemagglutinin-70 of clostridium botulinum and their application in a sandwich elisa.botulinum neurotoxins (bont) are produced by clostridium botulinum and cause severe neuroparalytic disease that if not treated quickly is often fatal. the toxin is produced as a 150 kda precursor protein (holotoxin) that is enzymatically cleaved to form two subunits, heavy and light chains, linked by a single disulfide bond. seven toxin serotypes are known. bont serotypes a1 and b1 are secreted as precursor toxic complexes (ptc) containing of the toxin and non-toxic associated proteins (naps) co ...201323600499
presence of clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism.clostridium botulinum is an anaerobic spore-forming bacterium prevalent in the environment, and causes botulism in man and animals via toxins. dairy cattle may be contaminated or infected by feed, water or other environmental factors. milk may also carry the pathogen. hence, milk and udder samples need to be tested. the number of clinical cases of bovine botulism in germany has been increasing since the mid-1990s. besides routine samples, additional 99 milk samples from 37 farms, and 51 udder sa ...201323585115
evaluation of the processing of perna perna mussels: the influence of water quality involved in the cooling operations in the physico-chemical and microbiological characteristics of the product.the state of santa catarina in brazil is a large producer of perna perna mussels. however, raw and processed mussels have a short shelf life because of their high microbiological count, such as vibrio spp. this study evaluated the microbiological and physicochemical quality of raw and ready-to-eat mussels and the quality of water and ice used in the processing of mussels.201323585097
alternative sigma factor sigk has a role in stress tolerance of group i clostridium botulinum strain atcc 3502.the role of the alternative sigma factor sigk in cold and osmotic stress tolerance of clostridium botulinum atcc 3502 was demonstrated by induction of sigk after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigk mutants under the respective conditions.201323563953
plasmid-borne type e neurotoxin gene clusters in clostridium botulinum strains.a collection of 36 clostridium botulinum type e strains was examined by pulsed-field gel electrophoresis (pfge) and southern hybridization with probes targeted to bote and orfx1 in the neurotoxin gene cluster. three strains were found to contain neurotoxin subtype e1 gene clusters in large plasmids of about 146 kb in size.201323563942
selective binding of 2'-f-c-di-gmp to ct-e88 and cb-e43, new class i riboswitches from clostridium tetani and clostridium botulinum respectively.c-di-gmp is a second messenger in bacteria and partly regulates bacterial physiology by binding to class i and ii riboswitches. four class i c-di-gmp riboswitch aptamer candidates, ct-e88, cb-17b, cb-e43 and cd-630 rnas, selected from a gemm rna sequence motif in the rfam database, were expressed and experimentally verified to bind to c-di-gmp. the two newly characterized c-di-gmp riboswitches, ct-e88 and cb-e43, bound c-di-gmp with nanomolar kd whereas the affinities of cb-17b and cd-630 for c- ...201323559271
detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms.in recent years biogas plants in germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. in particular, biogas plants are discussed to contribute to the spreading of escherichia coli infections in humans or chronic botulism in cattle caused by clostridium botulinum. metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at di ...201323557021
two-component signal transduction system cbo0787/cbo0786 represses transcription from botulinum neurotoxin promoters in clostridium botulinum atcc 3502.blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. whilst neurotoxin expression by the spore-forming bacterium clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor botr, have been implicated in this contr ...201323555260
development of a cell-based functional assay for the detection of clostridium botulinum neurotoxin types a and e.the standard procedure for definitive detection of bont-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (mba). the mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. cell-based assays provide an alternative to the mba in screening for bont-producing clostridia. here, we describe a cell-based assay utilizing a fluorescence reporter cons ...201323533420
structural insights into the functional role of the hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype c/d.botulinum neurotoxin (bont), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. produced by different strains of the anaerobic bacterium clostridium botulinum, bont effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. endocytosis, the first step of cellular intoxication, is triggered by the ~50 kda, heavy-chain receptor-binding domain (hcr) that is specific for a ganglioside ...201323523511
c3 exoenzyme lacks effects on peripheral axon regeneration in vivo.peripheral nerve injury triggers the activation of the small gtpase rhoa in spinal motor and peripheral sensory neurons. c3 transferase, an exoenzyme produced by clostridium botulinum that inactivates rhoa by adp-ribosylation, has been successfully applied in central nervous system (cns) lesion models to facilitate regeneration functionally and morphologically. until now it has not been demonstrated if c3bot exerts positive effects on peripheral axon regeneration as well. in organotypic spinal c ...201323521641
use of a new functional dual coating (fdc) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism.clostridium botulinum type a toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. the active dichain neurotoxin molecule is composed of a heavy chain (h-chain) of ~100 kda with the carboxy-terminal end consisting of a receptor-binding (hc) domain, while the amino-terminal (hn) domain is linked by a critical disulfide bond to a light chain (l-chain) of ~50 kda. although the mouse bioassay (mba) is traditionally used to confirm the presence of toxin in ...201323518650
genome sequence of the group iii clostridium botulinum strain eklund-c.the neurotoxins produced by clostridium botulinum strains are among the world's most potent toxins and are the causative agents of paralytic botulism. here, we present the draft genome sequence of the group iii c. botulinum strain eklund-c, including a pseudolysogen-like bacteriophage that harbors the type c neurotoxin operon.201323516187
molecular assembly of botulinum neurotoxin progenitor complexes.botulinum neurotoxin (bont) is produced by clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (pcs). the pcs are required for the oral toxicity of bont in the context of food-borne botulism and are thought to protect bont from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. the pc can differ in size and protein content depending on the c. botulinum strain. the oral toxicity of th ...201323509303
c2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein p53 into tumor cells.we have previously generated a recombinant c2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. a nontoxic moiety of clostridium botulinum c2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. in the present study, we used the c2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. the ...201323506195
multi-locus variable number tandem repeat analysis for clostridium botulinum type b isolates in japan: comparison with other isolates and genotyping methods.clostridium botulinum produces botulinum neurotoxin (bont) and causes botulism in humans and animals. recently, 15-loci multi-locus variable number tandem repeat analysis (mlva) for c. botulinum was developed for high-resolution and inter-lab comparative genotyping. this study examines the relation between mlva and other genotyping methods such as pulsed-field gel electrophoresis (pfge), multi-locus sequence typing (mlst), bont/b subtyping and bont/b gene location to evaluate mlva as a method ap ...201323499776
an evidence-based review of botulinum toxin (botox) applications in non-cosmetic head and neck conditions.botulinum toxin (botox) is an exotoxin produced from clostridium botulinum. it works by blocking the release of acetylcholine from the cholinergic nerve end plates leading to inactivity of the muscles or glands innervated. botox is best known for its beneficial role in facial aesthetics but recent literature has highlighted its usage in multiple non-cosmetic medical and surgical conditions. this article reviews the current evidence pertaining to botox use in the head and neck. a literature revie ...201323476731
effect of packaging systems and pressure fluids on inactivation of clostridium botulinum spores by combined high pressure and thermal processing.several studies have been published on the inactivation of bacterial spores by using high pressure processing in combination with heat. none of the studies investigated the effect of the packaging system or the pressurizing fluid on spore inactivation. the objective of this study was to select and validate an appropriate packaging system and pressure transfer fluid for inactivation of clostridium botulinum spores by using high pressure processing in combination with thermal processing. inactivat ...201323462082
conditions associated with clostridium sporogenes growth as a surrogate for clostridium botulinum in nonthermally processed canned butter.the objective of this study was to better understand the effect of butter composition and emulsion structure on growth and survival of clostridium sporogenes, used as a surrogate for c. botulinum in canned butter. the lack of a thermal process step in commercially available canned butter raises questions of potential safety, because it is hermetically sealed and generally exhibits anaerobic growth conditions, which are optimal for clostridium botulinum growth. without thermal processing, low-aci ...201323453518
phospholipase c produced by clostridium botulinum types c and d: comparison of gene, enzymatic, and biological activities with those of clostridium perfringens alpha-toxin.clostridium botulinum type c and d strains recently have been found to produce plc on egg yolk agar plates. to characterize the gene, enzymatic and biological activities of c. botulinum plcs (cb-plcs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. the enzymatic and hemolytic activities of the recombinant cb-plc were measured and compared with those of the clostridium perfringens alpha-toxin. each of the eight cb-plc g ...201323439504
recombinant streptavidin-c3bot for delivery of proteins into macrophages.we demonstrated previously that monocytes and macrophages are target cells for the rho-modifying clostridium botulinum c3 adp-ribosyltransferase. here, we report the construction, expression and characterization of a recombinant streptavidin-c3 fusion protein which allows for delivery of biotin-labelled molecules into the cytosol of macrophages via enzymatically inactive c3bot1e174q. the enzyme domain of diphtheria toxin was used as cargo to demonstrate proof of principle. this transport system ...201323422352
association of toxin-producing clostridium botulinum with the macroalga cladophora in the great lakes.avian botulism, a paralytic disease of birds, often occurs on a yearly cycle and is increasingly becoming more common in the great lakes. outbreaks are caused by bird ingestion of neurotoxins produced by clostridium botulinum, a spore-forming, gram-positive, anaerobe. the nuisance, macrophytic, green alga cladophora (chlorophyta; mostly cladophora glomerata l.) is a potential habitat for the growth of c. botulinum. a high incidence of botulism in shoreline birds at sleeping bear dunes national l ...201323421373
draft genome sequence of a clostridium botulinum isolate from water used for cooling at a plant producing low-acid canned foods.clostridium botulinum is a pathogen of concern for low-acid canned foods. here we report draft genomes of a neurotoxin-producing c. botulinum strain isolated from water samples used for cooling low-acid canned foods at a canning facility. the genome sequence confirmed that this strain belonged to c. botulinum serotype b1, albeit with major differences, including thousands of unique single nucleotide polymorphisms (snps) compared to other genomes of the same serotype.201323409268
glyphosate suppresses the antagonistic effect of enterococcus spp. on clostridium botulinum.during the last 10-15 years, an increase of clostridium botulinum associated diseases in cattle has been observed in germany. the reason for this development is currently unknown. the normal intestinal microflora is a critical factor in preventing intestinal colonisation by c. botulinum as shown in the mouse model of infant botulism. numerous bacteria in the gastro-intestinal tract (git) produce bacteriocines directed against c. botulinum and other pathogens: lactic acid producing bacteria (lab) ...201323396248
molecular dissection of botulinum neurotoxin reveals interdomain chaperone function.clostridium botulinum neurotoxin (bont) is a multi-domain protein made up of the approximately 100 kda heavy chain (hc) and the approximately 50 kda light chain (lc). the hc can be further subdivided into two halves: the n-terminal translocation domain (td) and the c-terminal receptor binding domain (rbd). we have investigated the minimal requirements for channel activity and lc translocation. we utilize a cellular protection assay and a single channel/single molecule lc translocation assay to c ...201323396042
notes from the field: botulism from drinking prison-made illicit alcohol - arizona, 2012.during november 24-27, 2012, the arizona department of health services (adhs) was notified that eight male inmates of prison a, a maximum security prison, had been hospitalized for treatment of an acute neurologic condition suspected to be botulism. botulism is a serious paralytic illness caused by a nerve toxin produced by the bacterium clostridium botulinum. all eight patients reported drinking pruno, an illicitly brewed alcoholic beverage that has been associated with botulism outbreaks in pr ...201323388552
foodborne botulism treated with heptavalent botulism antitoxin.to report a case of foodborne botulism and subsequent use of the investigational heptavalent botulism antitoxin (h-bat).201323362041
a recombinant fusion toxin based on enzymatic inactive c3bot1 selectively targets macrophages.the c3bot1 protein (~23 kda) from clostridium botulinum adp-ribosylates and thereby inactivates rho. c3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.201323349915
molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by clostridium botulinum hall a strain.seven distinct strains of clostridium botulinum (type a to g) each produce a stable complex of botulinum neurotoxin (bont) along with neurotoxin-associated proteins (naps). type a botulinum neurotoxin (bont/a) is produced with a group of naps and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. previous studies have indicated that bont/a complex composition is specific to the strain, the method of growth and the method of purification; conseq ...201323334849
efficacy and safety of a novel botulinum toxin type a product for the treatment of moderate to severe glabellar lines: a randomized, double-blind, active-controlled multicenter study.a new botulinum toxin type a (nbont) produced from the same strain of clostridium botulinum as onabotulinumtoxina (obont) is widely used in asia.201323301821
clinically and electrophysiologically diagnosed botulinum intoxication.in this case report, clinical and electrophysiological findings of 43-year-old female patient who developed clostridium botulinum intoxication after consumption of home-made canned food are presented. following the sudden onset of severe nausea and vomiting, diplopia, blurred vision, bilateral ptosis, weakness, speech and swallowing difficulties have developed and the patient declared that she has just tasted the canned beans after she had rinsed them several times. the case, where serological t ...201323291807
antagonistic effect of different bacteria on clostridium botulinum types a, b, d and e in vitro. 201323249773
structure-based drug discovery for botulinum neurotoxins.clostridium botulinum neurotoxin is the most poisonous substance known to humans. it is a potential biowarfare threat and a public health hazard. the only therapeutics available is antibody treatment which will not be effective for post-exposure therapy. there are no drugs available for post-intoxication treatment. accordingly, it is imperative to develop effective drugs to counter botulism. available structural information on botulinum neurotoxins both alone and in complex with their substrates ...201323239355
synchronized chaperone function of botulinum neurotoxin domains mediates light chain translocation into neurons.clostridium botulinum neurotoxin (bont) is a multidomain protein in which the individual modules work in synchronized cooperative action in order to enter into neurons and inhibit synaptic transmission. the di-chain protein is made up of the ~50 kd light chain and the ~100 kd heavy chain. the hc can be further subdivided into the n-terminal translocation domain (h(n)) and the c-terminal receptor binding domain (h(c)). bont entry into neurons requires the toxin to utilize the host cell's endocyto ...201323239351
assembly and function of the botulinum neurotoxin progenitor complex.botulinum neurotoxins (bonts) are among the most poisonous substances known to man, but paradoxically, bont-containing medicines and cosmetics have been used with great success in the clinic. accidental bont poisoning mainly occurs through oral ingestion of food contaminated with clostridium botulinum. bonts are naturally produced in the form of progenitor toxin complexes (ptcs), which are high molecular weight (up to ~900 kda) multiprotein complexes composed of bont and several non-toxic neurot ...201323239347
genetic diversity within clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (bonts) designated bont/a-g. this gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. early microbiological and biochemical characterization of c. b ...201323239346
improved detection methods by genetic and immunological techniques for botulinum c/d and d/c mosaic neurotoxins.clostridium botulinum type c and d strains produce serotype-specific or mosaic botulinum neurotoxin (bont). botulinum c/d and d/c mosaic neurotoxins (bont/cd and /dc) are related to avian and bovine botulism, respectively. the two mosaic bonts cannot be differentiated from authentic type c and d bonts by the conventional serotyping method. in this study, we attempted to establish novel methods for the specific detection of bont/cd or/dc. comparison with nontoxic component genes in type c and d s ...201323206412
outbreak of type c botulism in birds and mammals in the emilia romagna region, northern italy.over a 7-day period beginning 8 august 2011, a large number of wild birds of several species were found dead or with neurologic clinical signs along the shore of crostolo stream, in the emilia romagna region, italy. twenty-eight mallards (anas platyrhynchos), two hooded crows (corvus corone cornix), and three coypus (myocastor coypus) were found moribund on the crostolo stream bank, collected, and sent to istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna, reggio emilia ...201324502738
c3 rho-inhibitor for targeted pharmacological manipulation of osteoclast-like cells.the c3 toxins from clostridium botulinum (c3bot) and clostridium limosum (c3lim) as well as c3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the c3-catalyzed adp-ribosylation of rho results in inhibition of rho-signalling and characteristic morphological changes. since the fusion toxin c2in-c3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like raw 264.7 cells, its effects on raw 264.7-derived osteoclasts were ...201324386487
the effect of clostridium botulinum toxin type a injections on motor unit activity of the deep digital flexor muscle in healthy sound royal dutch sport horses.therapeutic reduction of the activity of the deep digital flexor (ddf) muscle may play a role in treatment of laminitic horses. clostridium botulinum toxin type a induces reduced muscle activity and has a spasmolytic effect in horses. in this study, the effectiveness of 200 iu c. botulinum toxin type a on reduction of ddf muscle activity was measured in seven healthy, sound, adult royal dutch sport horses. c. botulinum toxin type a was injected using ultrasound and electromyographic (emg) guidan ...201324360760
effect of clostridium botulinum toxin type a injections into the deep digital flexor muscle on the range of motion of the metacarpus and carpus, and the force distribution underneath the hooves, of sound horses at the walk.in the treatment of laminitis, reducing deep digital flexor muscle (ddfm) activity might diminish its pull on the distal phalanx, thereby preventing displacement and providing pain relief. injection of clostridium botulinum toxin type a into the ddfm of horses is potentially therapeutic. however, the effects of c. botulinum toxin type a on the gait characteristics of sound horses at the walk are not known. the aim of this study was to test if a reduced ddfm activity would lead to (1) alterations ...201324360731
a monoclonal antibody based capture elisa for botulinum neurotoxin serotype b: toxin detection in food.botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (bont), produced by the anaerobic bacterium clostridium botulinum. seven toxin serotypes (a-h) have been described. the majority of human cases of botulism are caused by serotypes a and b followed by e and f. we report here a group of serotype b specific monoclonal antibodies (mabs) capable of binding toxin under physiological conditions. thus, they serve as capture antibodies for a sandwich (capture) elisa. t ...201324253240
evaluation of dna extraction methods suitable for pcr-based detection and genotyping of clostridium botulinum.sufficient quality and quantity of extracted dna is critical to detecting and performing genotyping of clostridium botulinum by means of pcr-based methods. an ideal extraction method has to optimize dna yield, minimize dna degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin ...201323971807
management of animal botulism outbreaks: from clinical suspicion to practical countermeasures to prevent or minimize outbreaks.botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. the disease is caused by exposure to toxins produced by clostridium botulinum and other botulinum toxin-producing clostridia. botulism in animals represents a severe environmental and economic concern because of its high mortality rate. moreover, meat or other products from affected animals entering the food chain may result in a public health problem. to this end, early diagnosis is cruci ...201323971806
animal botulism outcomes in the anibiothreat project.botulism disease in both humans and animals is a worldwide concern. botulinum neurotoxins produced by clostridium botulinum and other clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the australia group list of biological agents. in 2010 the european commission (dg justice, freedo ...201323971804
rice hulls as a possible source of clostridium botulinum type c spores for poultry. 201324186999
ph responsive janus-like supramolecular fusion proteins for functional protein delivery.a facile, noncovalent solid-phase immobilization platform is described to assemble janus-like supramolecular fusion proteins that are responsive to external stimuli. a chemically postmodified transporter protein, dhsa, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic ph due to the iminobiotin linker and preserve the enzymatic activity ...201324156787
structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.botulinum neurotoxins (bonts) are produced by clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. bonts are released together with several auxiliary proteins as progenitor toxin complexes (ptcs) to become highly potent oral poisons. here, we report the structure of a ∼760 kda 14-subunit large ptc of serotype a (l-ptc/a) and reveal insight into its absorption mechanism. using a combination of x-ray crystallography, electron microscopy, and functional stu ...201324130488
femtogram-level detection of clostridium botulinum neurotoxin type a by sandwich immunoassay using nanoporous substrate and ultra-bright fluorescent suprananoparticles.we report a simple, robust fluorescence biosensor for the ultra-sensitive detection of clostridium botulinum neurotoxin type a (bont/a) in complex, real-world media. high intrinsic signal amplification was achieved through the combined use of ultra-bright, photostable dye-doped nanoparticle (dosnp) tags and high surface area nanoporous organosilicate (npo) thin films. dosnp with 22 nm diameter were synthesized with more than 200 times equivalent free dye fluorescence and conjugated to antibodies ...201323040876
two simultaneous botulism outbreaks in barcelona: clostridium baratii and clostridium botulinum.botulism is a severe neuroparalytic disorder that can be potentially life-threatening. in barcelona, spain, no outbreaks had been reported in the past 25 years. however, in september 2011, two outbreaks occurred involving two different families. a rare case of clostridium baratii which produced a neurotoxin f outbreak was detected in five family members who had shared lunch, and several days before that another family was affected by c. botulinum toxin a which was probably present in homemade pâ ...201323158693
occurrence of zoonotic clostridia and yersinia in healthy cattle.zoonotic pathogens are a frequent cause of disease worldwide. this study was designed to determine the occurrence of clostridium difficile, clostridium botulinum, and yersinia enterocolitica in cattle in southern bavaria, germany. the study population included 49 farms; 34 were dairy farms (30 also fattening beef cattle) and 15 were solely beef cattle farms. fecal and dust samples were collected from summer 2011 to summer 2012 and analyzed using a combination of enrichment procedures and real-ti ...201324112568
genetic characteristics of toxigenic clostridia and toxin gene evolution.clostridia comprise a heterogenous group of environmental bacteria containing 15 pathogenic species, which produce the most potent toxins. the origin of toxins is still enigmatic. it is hypothesized that toxins exhibiting an enzymatic activity have derived from hydrolytic enzymes, which are abundantly secreted by these bacteria, and that pore-forming toxins have evolved from an ancestor transmembrane protein. the presence of related toxin genes in distinct clostridium species and the variability ...201323707611
two multidrug-resistant clinical isolates of bacteroides fragilis carry a novel metronidazole resistance nim gene (nimj).two multidrug-resistant bacteroides fragilis clinical isolates contain and express a novel nim gene, nimj, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. hmw615, an appendiceal isolate, contains at least two copies of nimj on its genome, while hmw616, an isolate from a patient with sepsis, contains one genomic copy of nimj. b. fragilis nimj is phylogenetically closer to p ...201323716049
infant botulism: first two confirmed cases in slovenia and literature review.in europe, infant botulism is a rare but probably under-diagnosed disease. with the intent to spread the awareness of this potentially life-threatening disease, we present a review of the literature with the emphasis on european epidemiology and a practical approach to diagnosis. we also report the first two confirmed cases of infant botulism in slovenia and describe our way to the final diagnosis in a clinical setting where all appropriate diagnostic tests and treatment options are not readily ...201323707631
pathogenic psychrotolerant sporeformers: an emerging challenge for low-temperature storage of minimally processed foods.sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. in these products, the sporeforming pathogens of concern are bacillus cereus, bacillus weihenste ...201323536982
growth/no growth models for heat-treated psychrotrophic bacillus cereus spores under cold storage.the microbiological safety of refrigerated and processed foods of extended durability (repfed) is linked to spore-forming pathogens, more specifically clostridium botulinum and bacillus cereus. in this study two sets of growth/no growth (gng) models are presented for the spores of two b. cereus strains. the models incorporate both product (water activity (a(w)) and ph) and process parameters (pasteurization value at 90 °c (p(90)) or heating temperature). the first model evaluates the effect of f ...201323246607
the effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro.the use of glyphosate modifies the environment which stresses the living microorganisms. the aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. the presented results evidence that the highly pathogenic bacteria as salmonella entritidis, salmonella gallinarum, salmonella typhimurium, clostridium perfringens and clostridium botulinum are highly resistant to glyphosate. however, most of beneficial bac ...201323224412
[bioterrorism and pathogenic microorganisms].in recent years the use of pathogenic microorganisms in acts of bioterrorism has been the subject of major concern in many countries. this paper presents a possible application of viruses and bacteria for warfare and terrorist purposes, as well as a laboratory diagnosis to identify those agents. the viruses of smallpox (orthopoxvirus), of hemorrhagic fever and those belonging to filovirus have been highlighted, inter alia, as agents of human infection with bioterrorist intent. among the bacteria ...201324473660
mass spectrometric detection of protein-based toxins.this review focuses on mass spectrometric detection of protein-based toxins, which are among the most toxic substances known. special emphasis is given to the bacterial toxins botulinum neurotoxin from clostridium botulinum and anthrax toxins from bacillus anthracis as well as the plant toxin ricin produced by ricinus communis. a common feature, apart from their extreme toxicity, is that they are composed of 2 polypeptide chains, one of which is responsible for cell uptake and another that has e ...201323971809
cyclodextrin derivatives as anti-infectives.cyclodextrin derivatives can be utilized as anti-infectives with pore-forming proteins as the targets. the highly efficient selection of potent inhibitors was achieved because per-substituted cyclodextrins have the same symmetry as the target pores. inhibitors of several bacterial toxins produced by bacillus anthracis, staphylococcus aureus, clostridium perfringens, clostridium botulinum, and clostridium difficile were identified from a library of ∼200 cd derivatives. it was demonstrated that mu ...201324011515
genetic diversity of the flagellin genes of clostridium botulinum groups i and ii.botulinum neurotoxins (bonts) are produced by phenotypically and genetically different clostridium species, including clostridium botulinum and some strains of clostridium baratii (serotype f) and clostridium butyricum (serotype e). bont-producing clostridia responsible for human botulism encompass strains of group i (secreting proteases, producing toxin serotype a, b, or f, and growing optimally at 37°c) and group ii (nonproteolytic, producing toxin serotype e, b, or f, and growing optimally at ...201323603687
combined high pressure and thermal processing on inactivation of type a and proteolytic type b spores of clostridium botulinum.the aim of this study was to determine the resistance of multiple strains of clostridium botulinum type a and proteolytic type b spores exposed to combined high pressure and thermal processing and compare their resistance with clostridium sporogenes pa3679 and bacillus amyloliquefaciens tmw-2.479-fad-82 spores. the resistance of spores suspended in n-(2acetamido)-2-aminoethanesulfonic acid (aces) buffer (0.05 m, ph 7.0) was determined at a process temperature of 105°c, with high pressures of 600 ...201323905794
genomic and physiological variability within group ii (non-proteolytic) clostridium botulinum.clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. while studies have characterised variability between strains of group i (proteolytic) c. botulinum, the genetic and physiological variability and relationships between strains within group ii (non-proteolytic) c. botulinum are not well understood. in this study the genome of group ii strain c. botulinum eklund 17b (nrp) was sequenced and used to construct a whole gen ...201323679073
microbial contents of vacuum cleaner bag dust and emitted bioaerosols and their implications for human exposure indoors.vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. however, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. this study aimed to evaluate bioaerosol emission from various vacuum cleaners. we sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborn ...201323934489
gene expression profiling of clostridium botulinum under heat shock stress.during growth, c. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and ph change; however, its corresponding global transcriptional profile is uncharacterized. this study is the first description of the genome-wide gene expression profile of c. botulinum in response to heat shock stress. under heat stress (temperature shift from 37°c to 45°c over a period of 15 min), 176 c. botulinum atcc 3502 genes were differentially expressed. ...201324195079
application of high-density dna resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (bonts) that cause severe, potentially lethal intoxications in humans. these bont-producing bacteria are categorized in seven major toxinotypes (a through g) and several subtypes. the high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the bont components poses a great challenge for the screening and characterization of bont-producing strains.201323818983
multiplex real-time pcr for detecting and typing clostridium botulinum group iii organisms and their mosaic variants.botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. the disease in animals is mainly caused by toxins produced by clostridium botulinum strains belonging to group iii, although outbreaks due to toxins produced by group i and ii organisms have been recognized. group iii strains are capable of producing botulinum toxins of type c, d, and c/d and d/c mosaic variants. definitive diagnosis of animal botulism is made by combining clinical findings with l ...201323971808
biosynthesis of a thiamin antivitamin in clostridium botulinum.bacimethrin-derived 2'-methoxythiamin pyrophosphate inhibits microbial growth by disrupting metabolic pathways dependent on thiamin-utilizing enzymes. this study describes the discovery of the bacimethrin biosynthetic gene cluster of clostridium botulinum a atcc 19397 and in vitro reconstitution of bacimethrin biosynthesis from cytidine 5'-monophosphate.201424654570
climate change and infectious diseases in the arctic: establishment of a circumpolar working group.the arctic, even more so than other parts of the world, has warmed substantially over the past few decades. temperature and humidity influence the rate of development, survival and reproduction of pathogens and thus the incidence and prevalence of many infectious diseases. higher temperatures may also allow infected host species to survive winters in larger numbers, increase the population size and expand their habitat range. the impact of these changes on human disease in the arctic has not bee ...201425317383
gorilla gorilla gorilla gut: a potential reservoir of pathogenic bacteria as revealed using culturomics and molecular tools.wild apes are considered to be the most serious reservoir and source of zoonoses. however, little data are available about the gut microbiota and pathogenic bacteria in gorillas. for this propose, a total of 48 fecal samples obtained from 21 gorilla gorilla gorilla individuals (as revealed via microsatellite analysis) were screened for human bacterial pathogens using culturomics and molecular techniques. by applying culturomics to one index gorilla and using specific media supplemented by plants ...201425417711
clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. these gram-positive bacteria and their toxins include clostridium botulinum (c2 toxin), clostridium difficile (c. difficile toxin or cdt), clostridium perfringens (ι-toxin and binary enterotoxin, or bec), clostridium spiroforme (c. spiroforme toxin or cst), as well as bacillus cereus (vegetative insecticidal protein or vip). these gut-acting proteins for ...201425198129
development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library.botulinum toxins (bonts) are among the most toxic substances on earth, with serotype a toxin being the most toxic substance known. they are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by bont-producing clostridia. bont has been classified as a category a agent by the centers for disease control, and it is one of six agents with the highest potential risk of use a ...201424492304
vimentin mediates uptake of c3 exoenzyme.clostridium botulinum c3 exoenzyme (c3) selectively inactivates rhoa/b/c gtpases by adp-ribosylation. based on this substrate specificity c3 is a well-established tool in cell biology. c3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. based on different approaches vimentin was identified as membranous c3-interaction partner by mass spectrometry. vimentin in fact was partly localized at the outer surface of hippocampal ht22 cells and j744a.1 macrophages. doma ...201424967582
an electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of clostridium botulinum outbreak strains associated with type a botulism.specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (btas) such as bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin b (seb) and clostridial botulinum neurotoxins (bonts)). the present study describes an image-based 96-well meso scale discovery (msd) electrochemiluminescence (ecl) assay for simultaneous detection of btas in dairy milk products.201423873138
detection of anthrax and other pathogens using a unique liquid array technology.a bead-based liquid hybridization assay, luminex(®) 100™, was used to identify four pathogenic bacteria, bacillus anthracis, clostridium botulinum, francisella tularensis subsp. tularensis, and yersinia pestis, and several close relatives. hybridization between pcr-amplified target sequences and probe sequences (located within the 23s ribosomal rna gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. t ...201424147813
inhibitions of the translocation pore of clostridium botulinum c2 toxin by tailored azolopyridinium salts protects human cells from intoxication.c2 toxin from clostridium botulinum represents the prototype of clostridial binary actin adp-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. after receptor-mediated endocytosis of the c2 toxin complex, the binding/translocation component c2iia forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. the separate adp-ribosyltransferase component c2i translocates through this c2iia-pore ...201424394545
virulence plasmids of spore-forming bacteria.plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. unlike many other bacteria, the most common virulence factors encoded by plasmids in clostridium and bacillus species are protein toxins. clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as c. perfringens enterotoxin, epsilon-toxin, beta-toxin, and netb. ...201426104459
being prepared: bioterrorism and mass prophylaxis: part ii.although several biological agents have been recognized as presenting a significant threat to public health if used in a bioterrorist attack, those that are of greatest importance are known as the category a agents: bacillus anthracis (anthrax); variola major (smallpox); yersinia pestis (plague); francisella tularensis (tularemia); ribonucleic acid viruses (hemorrhagic fevers); and clostridium botulinum (botulism toxin). in the previous issue, part i of this review focused on the clinical presen ...201425356890
being prepared: bioterrorism and mass prophylaxis: part i.bioterrorism presents a real and omnipresent risk to public health throughout the world. more than 30 biological agents have been identified as possessing the potential to be deployed in a bioterrorist attack. those that have been determined to be of the greatest concern and possess the greatest potential of use in this arena are known as the category a agents: bacillus anthracis (anthrax); variola major (smallpox); yersinia pestis (plague); francisella tularensis (tularemia); viral hemorrhagic ...201425076398
tailored cyclodextrin pore blocker protects mammalian cells from clostridium difficile binary toxin cdt.some clostridium difficile strains produce, in addition to toxins a and b, the binary toxin clostridium difficile transferase (cdt), which adp-ribosylates actin and may contribute to the hypervirulence of these strains. the separate binding and translocation component cdtb mediates transport of the enzyme component cdta into mammalian target cells. cdtb binds to its receptor on the cell surface, cdta assembles and cdtb/cdta complexes are internalised. in acidic endosomes, cdtb mediates the deliv ...201425029374
cationic pamam dendrimers as pore-blocking binary toxin inhibitors.dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. here we identified poly(amido amine) (pamam) dendrimers as novel blockers for the pore-forming b components of the binary anthrax toxin (pa63) and clostridium botulinum c2 toxin (c2iia). these pores are essential for delivery of the enzymatic a components of the internalized toxins from endosomes into the cytosol of target cells. we demonstrate that at low μm concentration ...201424954629
immunological function of familial mediterranean fever disease protein pyrin.pyrin, encoded by mefv gene, is conserved in humans and mice. mutations in the mefv gene are associated with the human autoinflammatory disease familial mediterranean fever (fmf). pyrin can interact with the inflammasome adaptor asc and induce inflammatory caspase-1 activation in monocytic cells, but the physiological function of pyrin has been unknown for many years. here we summarize previous studies of pyrin function under the context of fmf and immunity, and discuss a recent study demonstrat ...201425307949
innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome.cytosolic inflammasome complexes mediated by a pattern recognition receptor (prr) defend against pathogen infection by activating caspase 1. pyrin, a candidate prr, can bind to the inflammasome adaptor asc to form a caspase 1-activating complex. mutations in the pyrin-encoding gene, mefv, cause a human autoinflammatory disease known as familial mediterranean fever. despite important roles in immunity and disease, the physiological function of pyrin remains unknown. here we show that pyrin mediat ...201424919149
infant botulism: is there an association with thiamine deficiency?infant botulism is an acute life-threatening condition and diagnosis is frequently delayed. therefore, the best time window to administer specific antibodies, at present the only etiology-based therapy, is often missed, entailing long periods of hospitalization in the picu. here we present a 3-month-old boy with infant botulism and respiratory failure, who quickly and favorably responded to thiamine supplementation. from the feces we isolated clostridium botulinum serotype a2. in addition to pro ...201425311602
evaluation of normalization reference genes for rt-qpcr analysis of spo0a and four sporulation sigma factor genes in clostridium botulinum group i strain atcc 3502.heat-resistant spores of clostridium botulinum can withstand the pasteurization processes in modern food processing. this poses a risk to food safety as spores may germinate into botulinum neurotoxin-producing vegetative cells. sporulation in bacillus subtilis, the model organism for sporulation, is regulated by the transcription factor spo0a and four alternative sigma factors, sigf, sige, sigg, and sigk. while the corresponding regulators are found in available genomes of c. botulinum, little i ...201424389585
molecular epidemiology of infant botulism in california and elsewhere, 1976-2010.infant botulism (ib), first identified in california in 1976, results from clostridium botulinum spores that germinate, multiply, and produce botulinum neurotoxin (bont) in the immature intestine. from 1976 to 2010 we created an archive of 1090 bont-producing isolates consisting of 1012 ib patient (10 outpatient, 985 hospitalized, 17 sudden death), 25 food, 18 dust/soils, and 35 other strains.201424924163
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